Figure 1.

Defective DNA damage-induced chromatin remodeling and ATM-Kap-1 signaling in Zmpste24−/− MEFs. (A) A representative gel photo of MNase accessibility assay. Arrows show ‘intact’ genomic DNA and oligonucleosomal DNA fragments. (B) Quantification of ‘intact’ genomic DNA in (A) by Image J®. (C) Representative immunoblots at various time points after 5 Gy of γ-irradiation. While the level of pS824-Kap-1 was significantly decreased upon γ-irradiation, pS473-Kap-1 was not obviously affected. At least three pairs of independently derived MEFs were examined. (D) Quantification of experiments in (C). Data represent mean ± SEM, n = 3. *P < 0.05. (E) Representative Western blotting in MNase-resistant fraction and total cell lysate upon γ-irradiation. (F) Representative immunobots in MEFs at 30 min after 5 Gy of irradiation. Data are representative of at least three independent experiments.

In response to DNA damage, ATM phosphorylates KAP-1 at Ser 824 (pS824-KAP-1), which weakens the binding of KAP-1 to MNase-resistant heterochromatin fraction and releases CHD3 from chromatin, leaving the heterochromatin de-condensed for loading essential repair proteins (Ziv et al., 2006; Goodarzi et al., 2008, 2011; Noon et al., 2010). To understand the mechanisms behind the defective chromatin remodeling in Zmpste24−/− MEFs, pS824-Kap-1 level was examined. The level of pS824-Kap-1 peaked around 30 min after γ-irradiation and was decreased gradually thereafter in wild-type cells (Fig. 1C,D), while it was significantly reduced in Zmpste24−/− MEFs. Consistently, in wild-type cells, the level of Kap-1 associated with MNase-resistant fraction was significantly reduced at 30 min after γ-irradiation in a dose-dependent manner, whereas it was hardly changed in Zmpste24−/− cells (Fig. 1E). As ATM is the only kinase responsible for pS824-Kap-1, and loss of ATM in MEFs also leads to defective chromatin remodeling upon DNA damage (Fig. S3), we asked whether ATM itself is affected in progeroid cells. Indeed, we found a significant reduction in the level of pS1981-ATM in Zmpste24−/− MEFs compared with wild-types (Fig. 1F and Fig. S4). Consistently, as a direct target of ATM (Shiloh, 2006), the level of pS343-Nbs1 was also significantly decreased in Zmpste24−/− MEFs in response to DNA damage (Fig. 1F and Fig. S4). Thus, these data indicate that defective ATM-Kap-1 signaling might underlie the defective chromatin remodeling in Zmpste24−/− MEFs.

Collectively, we found that the defective DNA repair in laminopathy-based progeria was attributable to compromised ATM-Kap-1 signaling and delayed global chromatin remodeling. Knocking down Kap-1 rescues the defective DNA repair and early senescence in progeroid cells, suggesting an important role of chromatin remodeling in laminopathy-based premature aging. The delayed yet completely relaxed chromatin in Zmpste24−/− MEFs implies the existence of a potential backup mechanism that mediates late chromatin remodeling in progeroid cells. Indeed, it has been reported that BRIT1 (BRIT-repeat inhibitor of hTERT expression), Brca1 and cofactor COBRA1, E2F1, and p53 regulate global chromatin relaxation/remodeling (Peng et al., 2009; Ye et al., 2001). This could be a backup mechanism regulating global chromatin remodeling in Zmpste24−/− MEFs, in response to the defective ATM-Kap-1 signaling.

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